Fibroblasts Can Be Genetically Modified to Produce Excitable Cells Capable of Electrical Coupling

Abstract

Background-Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions.Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. Methods and Results-High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (Ͼ80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation.

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy

“…1 In an earlier study, we demonstrated that fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. 2 In this study, fibroblasts were transduced with a MyoDencoding lentiviral vector to force differentiation into skeletal myotubes, resulting in the acquisition of an excitable phenotype. Gap junctional intercellular communication (GJIC) was achieved by transduction with a connexin43 (Cx43)-encoding vector.…”

Gene Therapy for Repair of Cardiac Fibrosis

“…In the study by Kizana et al, 22 no direct electrophysiological measurements were made. Action potentials were not recorded, and the proposed transmembrane currents were not demonstrated by patch-clamp techniques.…”

“…In cell cocultures, cardiomyocytes and fibroblasts can form functional gap junctions, 19,20 and recently, functional fibroblast-to-myocyte gap junctional coupling has also been demonstrated in the native rabbit sinoatrial node. 21 In this issue of Circulation, an article by Kizana et al 22 advocates the prospect that conduction defects in the heart might be repaired by genetic modification of fibroblasts. Human dermal fibroblasts were cultivated from pediatric skin samples and modified by lentivirus vector-mediated gene transfer to express MyoD, a skeletal myogenic determination factor.…”

“…24 In skeletal muscle as well, subthreshold depolarizations (to Ϫ60 mV) may already induce Ca 2ϩ release from the sarcoplasmic reticulum. 25 This is of interest because, in the experiments of Kizana et al, 22 a skeletal myogenic determination factor was used to induce myogenesis. To rule out the possibility that in modified fibroblasts, intracellular Ca 2ϩ transients are evoked without actually exciting the cell membrane, the membrane potential of these fibroblasts needs to be recorded.…”

Progress and prospects: Cell based regenerative therapy for cardiovascular disease

“…Efforts to manipulate cells ex vivo to direct them towards a desired phenotype are gaining momentum, and these will hopefully help to mitigate inflammation and Regenerative therapy for cardiovascular disease ED de Muinck et al scarring as well as the propensity for life threatening arrhythmia. 77,78 Homing and engraftment of circulating cells and the microenvironmental cues that dictate survival and proliferation of cells are areas that are anticipated to see a sustained scientific commitment, and as a result of this the application of implantable scaffolds to serve as an optimized microenvironment for cell survival, differentiation and proliferation may become an important adjunct to cardiac cell therapy. [79][80][81] …”